Production of vitamin b12 by agrobacterium radiobacter



United States Patent PRODUCTION OF VITAMIN B12 BY AGROBAC- TERIUM RADIOBACTER Johannes C. Hoogerheide, Whitefish Bay, Wis., assignor to Pabst Brewing Company, Milwaukee, Wis., a corporation of Delaware No Drawing. Application April 28, 1954, Serial No. 426,279

5 Claims. (Cl. 195-96) This invention relates to the production of vitamin B12, more particularly, to a microbial fermentation process for producing Vitamin B12. The invention is especially concerned with production of vitamin B12 by fermentation with a microorganism of the genus Agrobacterium.

It is known that vitamin B12 can be produced by fermentation using a variety of organisms and nutritive substances. As an illustration, U. S. Patent No. 2,595,499 discloses a number of classes of organisms and nutrients for the production of vitamin B12. U. S. Patent No. 2,561,364 refers, in particular, to the cultivation of F lavobacterium devomns in a suitable medium. U. S. Patent No. 2,576,932 discloses the use of Bacillus megatherium in a suitable medium.

It has now been discovered that vitamin B12 can be produced very advantageously by fermentation of a nutrient medium with a microorganism of the genus Agrobacterium of the family Rhizobiaceae.

Although the active substances produced are evaluated in terms of the equivalent amount of vitamin B12 and this terminology will be employed herein, the activity content may include also or in the alternative other compounds having vitamin B12 activity, as previously reported, which are meant to be included within the scope of the term.

In the invention, a nutrient medium is fermented with a vitamin Biz-producing strain of a microorganism of the genus Agrobacterium, preferably under submerged, aerated conditions and preferably also, with agitation. Fermentation is continued until the content of vitamin B12active substances is substantailly maximal, and a vitamin Biz-active concentrate is recovered from the fermentation product.

Organisms useful in the invention are described on pages 227231 of Manual of Determinative Bacteriology, Bergey, D. H. et al., (The Williams & Wilkins Company, Baltimore) 1948, 6th ed. Microorganisms which are particularly useful may be characterized by the following common properties. They are non-chromogenic, nonsporulating, small motile rods, are gram-negative and grow aerobically on most ordinary culture media. They all produce heavy slime in media containing carbohydrates. They do not produce gas nor appreciable acid from carbohydrates, nevertheless, such substrates are metabolized completely. Gelatin is not liquified, or only extremely slowly. The methyl red, Voges Proskauer and indole "ice reactions are negative. No appreciable growth is observed on Kosers citrate medium, unless a very heavy inoculum is used. Growth in milk produces a slight serum zone. They are non-pathogenic to plants and fail to produce root nodules on legume species.

Such strains, according to the present classification system of Bergeys manual, come under the genus Agrobacterium and, more specifically, fall within the species Agrobacterium radiobacter. Whereas, the description of this species as given in Bergeys manual represents the properties of certain typical standardstrains, it is a well recognized fact that there exists a marked variability in the properties of this organism. A few examples may sufiice.

Beyerinck, who first isolated and described this species, found that typical strains reduced nitrates and nitrites vigorously with production of gaseous nitrogen. In later years, however, strains were isolated which did not produce nitrogen gas from nitrate. The latter strains are at present recognized as typical in Bergeys manual, contrary to the original description of the species by Beyerinck.

The strains of Agrobacterium of the invention vary in their action on nitrate. Thus, some strains reduce nitrate with the production of nitrogen gas, others form no gas, but utilize all nitrate present as fast as the strains producing gaseous nitrogens, and again, others form nitrite from nitrate with a much slower nitrate reduction rate.

The strains of Agrobacterium useful in the invention also vary markedly in colony form on nutrient media. Some strains give colonies which are mucoid and viscous, while others produce colonies that are dry and rough. Often, the same strain may produce both types on the same nutrient agar, both types being equally good producers of the active substances.

While ability to grow at a pH of 10-11, the formation of hydrogen sulfide as determined by the Zobell method, browning on mannitol-calcium glycerophosphate and growth on Kosers uric acid medium have been described as characteristic of Agrobacterium radiobacter, a number of atypical strains do not possess all of these properties. Thus, reference to 'Agrobacteriurn and to the species radiobacter in the specification and claims is meant to include not only organisms having the characteristics given on pages 227-231 of Bergeys manual, but also atypical strains.

Several atypical strains which have characteristics differing in certain details from those given in the reference are further described in the table below. Rather than create new species for these strains, they are considered as atypical strains, not previously described, of the species Agrobacterium radiobacter. The table, in which characteristics of eight groups of Agrobacterium species are listed, illustrates the variations encountered by comparing the properties of two typical groups with six atypical groups. All strains in each group have all of the common characteristics of the genus Agrobacterium referred to above, and each produces an appreciable amount of vitamin B12, but the atypical strains differ in certain details from standard strains carried in stock collections.

Strain AIOO #4718 164* K300 5 382 5 255 280 B 263 157 Colonies on:

N utrlent agar Small col. no Same Large col. Same 3 Small col. Large col. Same Same.

slime. rnucold. no slime. mucoidfi /67,, Yeast extract-glu- Smooth, but Same Large mucoid Same 3 Mucoid Large mu- Same Same.

cose agar. not viscous. colJ' coid col.

Glycerol-agar 1 Viscous slimy White bu- Same 3 Same Smooth Same Viscous Same but tyrous scant slimy also buslimy. growth. transpartyrous col.

en Ca-gIyOero-phosphate White butyr- Same 1 Transparent White bu- Same I Transpar- White bu Transparagar 7 ans iridesmucoid. tyrous irient mutyrous lrient mucent. descent. coid. descent. eoid. Rose bengal agar Viscous slimy Semi-vis- Viscous trans- Same No growth. Same Small vis- Tiny transred col. cous red parent col., cous coloparent col.

col. no color abnics. (red).

sorption. Action on N itrate:

Nitrogen gas production.

Nitrite formation Nitrate utilized fast (less no.

than 5-7 days). Growth on Kosers:

Uricacidmedium same +(onlyaft- =l= or prolonged incubation).

H Production (Zo- No growth.

bells method). Growthwith heavy pelsame to+ +(ring +(rina liele on most liquid only). only).

nutrient media. Growth at pH=l0.5 Growth at pH=9.5 (6

days incubation) *Strains ATCC #4718 and #154 represent typical strains of Agrobacterium radiobacter. Strain ATOO #4718 is a strain of the species present in the American Type Culture Collection under N0. 4718. Strain #154 is an isolate from soil.

1 Some strains, otherwise identical, grow in colonies that are smooth but not viscous with a slight brown pigment excreted; others give transparent 'mucoid colonies.

2 Some strains, otherwise identical with #154, produce no nitrite and utilize nitrate much faster.

3 The same strain is often mixed with a variant, producing small non-viscous colonies.

4 Sometimes a slight trace of H28 is produced; some strains, otherwise identical with K300, fail to grow in this media.

5 A phage which lysis strain K800 also lysis strains #382 and #280, proving the close kinship of these strains Strains K300 and #382 seem to have the B Relatively poor growth except with heavy inoculurn.

' For composition of these media, see Hofer, J. Bact. 41, 193 (1941). 8 Method as recommended in Manual of Methods for pure culture study of bacteria (Soc. Am. Baeteriologists).

A number of strains of Agrobacterium are available pH is allowed to become too low, the growth of the orin the recognized culture collections. They may also be ganism is inhibited, and the yield of the desired end prodisolated from soil, for example, employing the Budinov- 40 not is thereby reduced. Bystryi technique (Mikrobiologiia, 10, 247-249, 1941), Fermentation is advantageously carried out at a tomthe method described by Smith (J. Bact. 15, 20, 1928), or perature of about 2540 (3., preferably about 28 C. similar methods. The strains are identified as described The inoculated medium is agitated and aerated, and above and tested for production of vitamin Biz-active fermentation is allowcd PFOCCed until the Optimum, substances, A i usually th case i i bi l th i usually the maximum, vitamin B12 production is achieved. not every strain of a given organism will produce vita- The fermentation product is then treated to produce i B ti substances d th quantities d d a concentrate of vitamin B12. Various methods for conwill vary, so th t i i necessary t l t a it i B centrating vitamin B12 are known to the art, several being producing strain, employing standard procedures and asdescribed in the Patents Previously identified For say m th d ample, liquid may be removed from the fermentation The fermentation medium may have the usual combroth by evaporation. which y be preceded y fi ponents employed for i Purpose, e a source f tion. Or the broth may be filtered and contacted with i il bl nitrogen, h as a proteinaceous substance, activated charcoal or fullcrs earth to adsorb the active at source of assimilable carbon, such as a carbohydrate, Substances thereon The rfisulfing Vitamin BIZ-active and trace elements, particularly Cobalt, Among the concentrates are useful as animal food supplements. Alteinaceous and carbohydrate materials which can be used further concentration may be efiected, Such as by are yeast extracts, casein digests, meat extracts, steep elutlon from the adsorbent P cPncfmtration 0f the liquors, distillers solublcs, soya, glucose, sucrose, molasses eluate: followed by further punficallon 1f 'f and a great'variety of similar substances. Suitable media T followlng examples are ,furmshed 9 assls? in P are illustrated in the patents previously referred to. Y q a Complete understandms of t {nVentwI}, P A sounce of cobalt providing an addition of about 0.1 1t 15 to be understood h F mYentlon 15 hmlted to 20 parts per million of cobalt to the nutrient medium thereto nor to the speclfic mgrecllents: P PP and is f bl incorporated in the medium. as described procedures set forth therein, wh1cl1 are given only for in U. S. Patent No. 2,595,499. It is further preferred Purposes of illustratlon' to supply the cobalt in the form of a cobalt salt soluble in the medium, such as cobalt nitrate, cobalt sulfate and the like, although it may be supplied in other forms. Media were prepared containing 1% meat extract, 1% The pH of the fermentation is preferably within the corn steep solids, 1% dextrose, 3% sucrose and 15 range of about 6.5 to 8.2, the optimum usually being p. p. m. of cobalt as CoSO4.7H2O. Forty-milliliter porabout 7.5. The pH may be controlled by adding dextions of these media were adjusted to pH 7.5 and placed Example 1 trose, sucrose or similar substances, by the addition of in 250 ml. Erlenmeyer flasks. The flasks and their conmineral acids or by adding ammonium hydroxide. If tents were sterilized for 15 minutes at 121 C. After the pH is allowed to become too high, the higher alkasterilization, the flasks were cooled and the contents inlinity tends to decompose the desired end product or preoculatedwith 2 ml. from a 24 hour liquid culture of an ventits formation and thereby decrease the yield. If the Agrobacterium species.

The inoculated flasks were incubated at 28 C. for four days on a rotary shaker, fermentation thus being carried out under submerged, agitated and aerated conditions. Upon completion of the incubation period, the vitamin B12 content of the fermentation broths was determined by a modification of the proposed USP method (U. S. Pharnr, XlV, third Suppl, 15-19) using Lactobacz'llus lez'clzmtmnii ATCC 7830 as the test organism. The average vitamin B12 content of the broths was found to be 0.81 micrograms per gram of broth.

Example 2 The procedure of Example 1 was repeated, except that the flask contents were inoculated with 2 ml. from a 24 hour culture of Agrobacterium variant K300. The average vitamin B12 content of the fermentation broths was 0.715 microgram per gram of broth.

For the production of a vitamin B12 concentrate, the pH of the fermentation broth may be adjusted to pH. 4.5-5.0 by addition of hydrochloric acid. The fermentation broth is heated to about 70 C. for 15 minutes, in order to liberate the vitamin from the cells. The culture, thus treated, may now be concentrated under vacuum and drum dried, or it may first be filtered for removal of cell debris, then concentrated under vacuum and drum dried.

Example 3 The procedure of Example 1 was replaced except that the medium used had the following composition: 3% cornsteep liquor solids, 1%% dextrose and 15 p. p. m. of cobalt as COSOAJHZO. The culture was inoculated with 2 ml. of a 24 hour culture of Agrobacterium variant K300 and incubated at 28 C. for four days on a rotary shaker. The vitamin B12 content of the fermentation broth at the end of the fermentation period was found to be 1.85 micrograms per gram of broth.

Example 4 The procedure of Example 3 was repeated, except that the flask contents were inoculated with 2 ml. of a 24 hour culture of Agrobacterium radiobacter A. T. C. C. 4718, a strain maintained in the collection of the American Type Culture Collection. The average vitamin 1312 content of the fermentation broths was 0.75 microgram per gram of broth.

Example 5 The procedure of Example 3 was repeated, except that the flask contents were inoculated with 2 ml. of 24 hour cultures of strains #154, #382, #255 and #263, respectively. Strain #154 is a typical strain of Agrobacterium radiobacter, isolated from soil, whereas strains #382, #255 and #263 are atypical strains of this species. The

6 average vitamin B12 content per gram of broth of th fermentation broths after 4 days incubation was as follows: 0.6 microgram B12 for strain #154, 1.5 microgram B12 for strain #382, 1.05 microgram B12 for strain #255 and 2.1 microgram B12 for strain #263.

Example 6 The procedure of Example 1 was repeated except that the medium used had the following composition: 3% cornsteep liquor, 1 /z% dextrose, 1% meat extract and 15 p. p. m. of cobalt as CoSOtJHzO. The culture medium was inoculated with 2 ml. of 24 hour cultures of Agrobacterium variants #255, #382 and K300, respectively. The average vitamin B12 content per gram of broth of the fermentation broths was 1.2 micrograms for strain #255, 1.5 micrograms for strain #382 and 1.9 micrograms for strain R300.

The invention thus provides a very advantageous process for producing vitamin B12 in good yields, increasing its availability and serving to meet the large demand for the vitamin for nutritional and therapeutic purposes.

The invention is hereby claimed as follows:

1. The process which comprises fermenting a nutrient medium with a vitamin Biz-producing strain of Agrobacterium radiobacter under submerged aerated conditions.

2. The process which comprises fermenting a nutrient medium with a vitamin Biz-producing strain of Agrobacterium rcdiobacter until the vitamin B12 content is substantially maximal.

3. The process which comprises fermenting a nutrient medium containing a source of assimilable nitrogen, a source of assimilable carbon and a source of cobalt with a vitamin Bizroducing strain of Agrobacterium radiobacter.

4. The process which comprises fermenting a nutrient medium with a vitamin Biz-producing strain of Agrobacterium radiobacter to produce a fermentation product containing vitamin B12.

5. The process which comprises fermenting a nutrient medium with a vitamin Biz-producing strain of Agrobacterium radiobacter, and producing a concentrate of vitamin B12 from the fermentation product.

References Cited in the file of this patent UNITED STATES PATENTS Wood et a1 May 6, 1952 Rickes Mar. 1, 1955 OTHER REFERENCES 

1. THE PROCESS WHICH COMPRISES FERMENTING A NUTRIENT MEDIUM WITH A VITAMIN B12-PRODUCING STRAIN OF AGROBACTERIUM RADIOBACTER UNDER SUBMERGED AERATED CONDITIONS. 